Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing

Conventional culture methods were compared to a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 from enrichment cultures of various types of artificially inoculated and naturally contaminated foods. The multiplex PCR assay was evaluated in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen for simultaneous detection. The sensitivity of the assay was 相似文献   

2005年石家庄市部分食品中产单核细胞李斯特菌污染状况调查

[目的]了解石家庄市主要食品中产单核细胞李斯特菌污染情况，为预防和控制可能由产单核细胞李斯特菌污染引发的食源性疾病提供依据。[方法]2005年，在石家庄市采集市售5类食品进行分离鉴定。[结果]检测5类250份食品样品，检出13株产单核细胞李斯特菌，阳性率为5．20％。130份生肉类阳性率为6．92％（猪肉为20．00%，羊肉、牛肉均为3．33％，鸡肉为2．50％；30份熟肉制品阳性率为6．67％，30份生食蔬菜阳性率为6．67％；36份水产品、24份奶粉均为阴性。13株产单核细胞李斯特菌溶血试验均阳性。[结论]生肉类、熟肉制品、生食蔬菜是主要的危险食品。     

Taq Man-MGB探针Real-timePCR快速检测单增李斯特菌的研究

目的：建立TaqMan—MGB探针Real—time荧光PCR快速检测单增李斯特菌技术。方法：在对单增李斯特菌invA序列进行分析、比较基础上，设计一对特异性TaqMan—MGB探针法引物及探针，通过Real—timePCR反应条件和反应体系的优化，实现对单增李斯特菌的快速检测；用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果：方法的灵敏性高，其循环阈值与模板浓度的对数值具有很好的对应关系，最低可检测57个拷贝数，经18h增菌，可检测低至4．5cfu／ml的细菌；特异性强，检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值（cT值）均小于25，而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值（CT值）大于35；快速，最快1h可出结果，实际样品20h可出结果，而常规细菌培养法需要一周以上；对103份速冻米面食品进行检测，13份阳性，与常规方法无显著性差异（P〉0．05）。结论：TaqMan—MGB探针的单增李斯特菌Real—time荧光PCR检测方法具有特异性强，敏感性高，易操作等优点，可推广应用。     

Development and evaluation of a real-time polymerase chain reaction assay targeting iap for the detection of Listeria monocytogenes in select food matrices

Listeria monocytogenes is an intracellular foodborne pathogen that has been associated with severe human illnesses. Various rapid detection methods have been developed for the specific detection of this pathogen. In the present study, a real-time quantitative polymerase chain reaction (PCR) assay targeting iap, a gene encoding extracellular protein p60, was developed for L. monocytogenes. The PCR efficiency is above 85% and the limit of detection (LOD) is 30 copies of genome per reaction for all strains tested. The assay exhibited 100% inclusivity and exclusivity rates. The detection of L. monocytogenes in five food matrices, whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts, was evaluated with and without enrichment. Without enrichment, the LOD for all food matrices were 4×10(3)?CFU/mL food enrichment mix for whole milk and 4×10(4)?CFU/mL for all other foods. With 24?h incubation in Buffered Listeria Enrichment Broth, the LOD was 3?CFU/25?g food for whole milk, turkey deli meat, and smoked salmon and 9?CFU/25?g food for soft cheese and alfalfa sprouts. With 48?h incubation, the LOD was 3?CFU/25?g food for all matrices. This quantitative PCR appears to be a promising alternative for rapid detection of L. monocytogenes in select foods.     

Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples

Use of universal pre-enrichment broth (UPB) as a primary enrichment medium for detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples was evaluated. There were no differences in bacterial growth between UPB and selective primary enrichment broths for each pathogen inoculated individually or in combination at 10(1) and 10(2) colony forming units/mL. In addition, no differences were observed when UPB and selective primary enrichment broths were compared for detection efficiency of pathogens in artificially contaminated raw milk and fecal samples. Listeria enrichment broth (LEB) was compared with UPB to support growth of L. monocytogenes from naturally contaminated environmental samples. Listeria monocytogenes was isolated from seven of 30 samples enriched in UPB and six of 30 samples enriched in LEB. Dairy farm environmental samples were examined for recovery of the three pathogens using UPB. Subsequent isolation was achieved using selective secondary enrichment of each pathogen. Listeria monocytogenes, Salmonella spp., and E. coli O157:H7 were isolated in 13.4% (30 of 224), 8.9% (20 of 224), and 2.2% (five of 224) of samples, respectively. Isolation rates of the three pathogens were somewhat higher than in previous reports. Overall, UPB supported growth of test pathogens to detectable levels within 24 h. Our results demonstrate that UPB has potential for routine use in isolation of foodborne pathogens from diverse environmental samples.     

Prevalence and serotypes of Listeria monocytogenes contamination in Chinese beef processing plants

The aim of this work was to study the epidemiology of Listeria spp., particularly Listeria monocytogenes, and to identify the serotypes present in contaminated samples from beef processing plants in China. A total of 439 samples were obtained from bovine feces, hides, and carcasses at three commercial processing plants. A standard protocol (ISO 11290-1) was followed to detect Listeria spp. and L. monocytogenes, and multiplex polymerase chain reaction was used to identify the various L. monocytogenes serotypes. The overall prevalences of Listeria spp. and L. monocytogenes were 65.6% and 26.4%, respectively, and the contamination was highest in the hide samples. The identified L. monocytogenes serotypes were 1/2c and 1/2a. The results of the current study indicate that Listeria spp. contamination is common in Chinese beef processing plants; specific measures should be taken to prevent and/or treat L. monocytogenes contamination of feces and hides in beef slaughter plants. Furthermore, because Listeria spp. contamination was found to be prevalent, it should, therefore, be studied further. The prevention of cases of sporadic listeriosis in China should also be addressed.     

深圳市熟肉制品致病菌监测结果分析

目的:了解深圳市熟肉制品受致病菌污染的状况,为食源性疾病的预防和控制提供科学依据。方法:对酒店、超市和市场禽类熟肉制品随机采集360份样品,依据国标方法、荧光PCR方法及全自动微生物分析仪,对上述样品进行致病菌检测。结果:360份样品共检出致病菌38株,检出率10.56%,其中沙门菌4株、金黄色葡萄球菌32株、单增李斯特菌2株。酱卤类、烧烤类、白切类熟肉制品检出率分别为11.66%、3.33%、16.67%。结论:深圳市熟肉制品受致病菌污染严重,以金黄色葡萄球菌、沙门菌和单核李斯特菌污染为主;白切类熟肉制品受致病菌污染最为严重为16.67%;其次是酱卤类为11.66%;最后是烧烤类为3.33%。说明酒店、超市熟肉制品存在很大问题,应加大对熟肉制品监管力度,保证食品质量,确保消费者健康。     

Prevalence and risk factors for Listeria monocytogenes in broiler flocks in Shiraz, southern Iran

Listeria monocytogenes has been identified as an important foodborne pathogen in recent years. In humans, it most commonly affects pregnant women, neonates, children, elderly people, and persons with a suppressed immune system. It could contaminate both raw and cooked meat and poultry products. Studies regarding prevalence and risk factors of L. monocytogenes in broilers flocks are limited. Therefore, the present study was conducted to determine the prevalence and risk factors for L. monocytogenes in poultry flocks in Shiraz, southern Iran. During August to September 2009, in total, 100 broiler flocks were selected at slaughter, and 21 specimens were collected from cloacal samples from each flock. Polymerase chain reaction (PCR) was performed on the samples enriched in buffered Listeria enrichment broth (BLEB), using specific primers. Furthermore, enriched samples in BLEB and/or BLEB treated with 5% KOH were subcultured on Palcam medium. Data about farm and flocks were collected using a structured questionnaire. The prevalence of L. monocytogenes was 7% (95% CI, 2-12%) and 1% using PCR and culture, respectively. Results showed that using antibiotics during rearing period was dramatically reduced the rate of isolation (odds ratio [OR]=0.07, p=0.03), whereas house capacity of more than 10,000 birds (OR=24.03, p=0.04) and number of houses (OR=2, p=0.02) significantly increased the prevalence. The correlation between poor management of large poultry flocks and increasing the risk of contamination was more likely due to the recontamination of cooked poultry/undercooking or cross-contamination of other ready-to-eat foods.     

食品中单核细胞增生李斯特菌污染及耐药状况调查

目的:了解吉林省市售八类食品中单增李斯特菌的污染状况和耐药现状。方法:采集市售八类食品样品,采用显色培养基,API及BAX分离鉴定,K-B法药敏实验。结果:1251份样品共检出单增李斯特菌74株,总检出率为5.9%。主要在熟肉制品(17.0%)和凉拌菜(9.1%)。74株菌株对氨苄西林、青霉素、庆大霉素、卡那霉素全部敏感。头孢噻肟、新霉素耐药率达50%以上。其次为恩氟沙星(35.1%)、环丙沙星(33.8%)和诺氟沙星(25.6%)。结论:吉林省市售八类食品中尤其是熟肉制品和凉拌菜中单增李斯特菌污染和耐药现象严重。应高度重视并加强污染状况及耐药性监测,保证食品安全和人类健康。     

莒南县食品中单核细胞增生李斯特菌污染状况调查

目的掌握莒南县食品中单核细胞增生李斯特菌污染状况。方法按GB4789．30—2003方法分离单核细胞增生李斯特菌。结果2006年从610份食品中共分离到单核细胞增生李斯特菌39株，总污染率为6．32％，生肉类、熟肉及生食蔬菜的污染率分别为9．94％、4．48％、3．78％。结论该县存在发生李斯特菌食物中毒的潜在危险。     

2000～2004年浙江省食品中产单核李斯特菌污染状况调查

目的：掌握浙江省食品中产单核李斯特菌污染状况。以及内化素基因（Inl）和李氏溶血素O（Hly）两种致病基因携带情况。方法：按GB4789．30方法分离单增生李斯特菌，采用PCR技术检测李斯特菌的两种致病基因。结果：2000～2004年从2282份食品中共分离产单核李斯特菌163株，总污染率为7．14％（163／2282）。生肉类、熟肉及水产品的污染率分别为12．80％、7．67％和2．24％；114份生食蔬菜中1份标本分离到产单核李斯特菌；285份乳及乳制品、59份冰激凌均未分离到产单核李斯特菌；163株产单核李斯特菌有155株同时检测到内化素基因和李氏溶血素O基因。结论：我省存在发生李斯特菌食物中毒的潜在危险。     

Listeria spp. in the coastal environment of the Aqaba Gulf, Suez Gulf and the Red Sea

Listeria monocytogenes is an important pathogen which causes an infection called listeriosis. Because of the high mortality rate (~30%) associated with listeriosis, and the widespread nature of the organism, it is a major concern for food and water microbiologists since it has been isolated from various types of foods, including seafood, as well as from the aqueous environment. To investigate the prevalence of this pathogen in the Aqaba Gulf (12 sites), Suez Gulf (14 sites) and Red Sea (14 sites), 200 water samples (collected during five sampling cruises in 2004), 40 fresh fish samples and 15 shellfish samples were analysed using the enrichment procedure and selective agar medium. All water samples were also examined for the presence Listeria innocua which was the most common of the Listeria spp. isolated, followed by L. monocytogenes, with a low incidence of the other species. During the whole year, the percentage of Listeria spp. and L. monocytogenes in 200 water samples was 20.5% (41 samples) and 13% (26 samples) respectively. In fresh fish (40 samples) it was 37% (15 samples) and 17.3% (7 samples) and in shellfish (15 samples) 53% (8 samples) and 33% (5 samples) respectively. In water samples, there was an association between the faecal contamination parameters and the presence of the pathogen; however, water salinity, temperature, dissolved oxygen and pH did not influence the occurrence of this bacterium. These results may help in the water-quality evaluation of the coastal environments of these regions.     

食品中单增李斯特菌PCR检测方法建立与评价

目的建立单核细胞增生李斯特菌（Listeria monocytogenes，LM）快速、敏感、特异的PCR诊断方法。方法采用聚合酶链式反应技术（PCR）特异性扩增单核细胞增生李斯特菌溶血素基因（hlyA），并评价该方法的特异性与敏感性。结果在706bp处出现nuc基因的目的片断，只有单增李斯特菌的目的片段获得扩增，其他菌种扩增均呈阴性；该方法可以检测到3．3ng／L的DNA。结论PCR方法比传统细菌检测方法更特异、快速、灵敏和简便。为食品中单增李斯特菌的快速检测提供了新的手段。     

呼和浩特市食品中单核细胞增生李斯特氏菌污染状况调查

目的本次研究旨在通过对呼市居民主要的消费食品进行单增李斯特菌的污染调查,从而掌握该菌对食品的污染情况。方法随机抽取超市、农贸市场、酒店、食品店出售的食品共计15种543份,采用国标法进行检测。结果 543份食品中,4种食品中检测出6株单增李斯特菌,检出率为1.11%。其中生畜肉检出2株,检出率3.77%;生禽肉检出2株,检出率6.67%;凉拌菜检出1株,检出率3.33%;即食非发酵类豆制品1株,检出率1.82%,其余食品均未检出。结论生畜肉、生禽肉、凉拌菜、即食非发酵类豆制品检出率均较高。提示存在发生食源性单增李斯特菌病的潜在危险。因此,加强食品的卫生监督管理,开展食品污染源调查,提高公众在食品储藏和加工方面的卫生意识十分必要。     

单增李斯特菌PCR-ELISA快速检测技术研究

目的建立快速检测单增李斯特菌的PCR-ELISA方法,将其应用于人工污染的食物样品检测。方法根据GenBank数据库资料,应用分子生物学软件DNAMAN6.0和Primer Premier5.0,以单增李斯特菌的致病基因hlyA为基础,选用PCR引物,应用地高辛标记试剂盒,获得单增李斯特菌特异的地高辛标记片段。根据PCR目的片段序列,设计特异性捕获探针,建立了单增李斯特菌PCR-ELISA快速检测方法。应用该方法对不同血清型的单增李斯特菌食品分离株进行了检测,并将PCR方法与PCR-ELISA方法对人工污染单增李斯特菌的牛奶样品的检测敏感性进行了比较。结果应用单增李斯特菌特异的PCR-ELISA方法完成检测约需6h。该方法对单增李斯特菌分离株检测的结果,与国家食源性疾病检测网的鉴定结果100%符合。经过12h的增菌培养,PCR-ELISA方法最低可从25ml样品中检出1CFU,检测敏感性为传统PCR方法的10～100倍。结论建立了单增李斯特菌PCR-ELISA快速检测方法。该方法敏感性高、特异性强、可靠性好,对于提高食源性疾病预警、预测能力,增强检测的时效性和准确性,具有推广应用价值。     

食品中沙门菌特异性二重聚合酶链反应检测方法的研究

目的建立二重聚合酶链反应(PCR)方法快速检测食品中的沙门菌(Salmonella spp.)。方法以沙门菌特异性基因invA、hilA为靶基因,选择2对引物对16株沙门菌和24株非沙门菌进行扩增,验证二重PCR的特异性。梯度稀释DNA,以不同稀释度的DNA PCR扩增。在猪肉中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增。应用该方法检测实际样品。结果以invA、hilA为靶基因的两对引物对沙门菌的检出均有很好的特异性,PCR能检出0.1332pg的沙门菌DNA,人工污染猪肉的检测限为2.5cfu/ml。本研究共检测了53份食品样品,有21份检出了沙门菌。结论建立了适用于肉类、水产品及蛋糕等食品中的沙门菌检测的快速、灵敏、特异的二重PCR方法。     

湖州市食品中单增李斯特菌的污染状况调查

目的:了解湖州市食品中单增李斯特菌的污染状况。方法:按国标方法,采用进口显色培养基,对食品进行单增李斯特菌的分离、生化鉴定。结果:281份样品中共检出22株,污染率为7.83%。其中冷冻/藏鸡肉中污染率最高,为25.00%。结论:湖州市食品中单增李斯特菌的污染比较严重,应加强对生肉制品等的监督管理,以防食源性疾病的发生。     

改良分子信标-实时PCR快速检测产单核李斯特菌

目的：建立改良分子信标-实时PCR检测产单核李斯特菌（LMO）的快速方法，应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法：根据GenBank公布的LMO hlyA基因的保守序列，设计引物和改良分子信标探针，建立改良分子信标-实时PCR检测体系，应用于食品中LMO检测。结果：改良分子信标-实时PCR反应体系DNA灵敏度为110fg，菌液灵敏度为99cfu／ml或4cfu／PCR反应体系，无交叉反应。以此反应体系检测28株LMO，均出现特异的荧光信号。上述方法可将检测时问由原来的至少4d缩短至1d。对228份食品进行LMO检测，8份增菌液LMO实时荧光PCR阳性，其中6份1310细菌培养阳性。结论：改良分子信标-实时PCR反应体系快速、灵敏度高，特异性强．能提高LMO的检出率和准确性，可应用于LMO食品污染状况调查及食物中毒的快速诊断。     

Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction

Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.     

深圳市东门菜肉市场单核细胞增生李斯特氏菌污染状况分析

目的通过对深圳市东门菜肉市场单核细胞增生李斯特氏菌污染状况的调查,加大对冷藏食品中威胁人类健康单核细胞增生李斯特氏菌的重视。方法根据中华人民共和国国家标准食品卫生微生物学检验总则单核细胞增生李斯特氏菌检验(GB4789.30-2003)、荧光PCR法。结果对所采的50份样品用国标方法和荧光PCR法均检出3株单核细胞增生李斯特氏菌。结论深圳市东门菜肉市场存在着单核细胞增生李斯特氏菌污染,本次调查的污染率为6%,建议管理部门和卫生部门加大卫生监督力度,预防单核细胞增生李斯特氏菌食物中毒的发生。